Organophosphorus (OP) nerve agents are classified as neurotoxins as they serve as potent inhibitors of acetylcholinesterase (AChE). Inhibition of AChE leads to toxic accumulation of acetylcholine in the synapse and neuromuscular junctions, resulting in hyper-stimulation of these pathways. OP nerve agents represent a significant threat and thus, there is an urgent need to develop therapeutic treatments for those exposed or poisoned by OPs. Phosphotriesterase (PTE) hydrolyzes a broad range of substrates including OPs. However, its activity varies considerably from substrate to substrate and possesses a short half-life, in vitro. Recently, we developed a fluorinated PTE variant via computational methods that exhibits superior stability and half-life. Our long-term goal is to develop robust, PTE-based therapeutics for treatment of V-agent (an OP nerve agent) exposure. Our central hypothesis is that combining non-canonical amino acid (NCAA) incorporation into PTE for increased stability with Rosetta-based computational design can lead to the development of highly stable and active fluorinated PTE variants for VX, VR and VM. Our rationale is that once we identify fluorinated PTE variants active for V-agents, they can be tested in vivo for therapeutic efficacy as a novel treatment for OP exposure. Our specific aims focus on employing Rosetta to design fluorinated PTE variants for enhanced hydrolysis of VX, VR, and VM with improved half-life and stability (aim 1) and learning from experimental results to design and investigate a second generation set of fluorinated PTE2 variants with improved function for the V agents (aim 2). The proposed studies will have an important translational impact on therapeutic interventions for OP poisoning, specifically those exposed to V agents. Our combined biosynthetic and computational approaches for NCAA-bearing enzymes are expected to advance the design of therapeutic enzymes and treatment of exposure.